As a result of development in gene recombination technology, it is now possible to manufacture industrially useful proteins in large amount utilizing prokaryotes and eukaryotes as hosts. As a host of prokaryote, bacterium such as Escherichia coli is commonly used while, as a host of eukaryote, yeast such as genus Saccharomyces or Pichia pastris is commonly used.
Since yeasts usually grow quickly, they can be cultured in higher cell density than bacteria. Further, in yeasts, separation of cells from culture liquid is easier than in bacteria whereby extracting and purifying steps of the produced protein can be made simpler. Therefore, yeasts have now been frequently used as the hosts for the production of useful proteins by means of gene recombination technology.
In view of usefulness of the yeasts as such, the Applicants have selected, from various yeasts, the Cryptococcus sp. S-2 strain [this is a kind of basidiomycetous yeast and has been internationally deposited on Sep. 5, 1995 under the accession number FERM BP-10961 at International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (current name: National Institute of Technology and Evaluation) located at Central No. 6, 1-1 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Postal Code: 305-8566) (which has now been moved to Room 120, 2-5-8 Kazusa Kamatari, Kisarazu-shi, Chiba-ken, Japan (Postal Code: 292-0818)] exhibiting a characteristic of producing the proteins such as α-amylase, acidic xylanase and cutinase in large amount and of secreting them to the outside of cells (See Non-Patent Document 1).
The Applicants have further proposed the large-amount production of heterologous proteins other than the proteins which are inherently produced by the yeast, utilizing the high productivity of extracellular proteins by the above strain. Furthermore, in order to more efficiently select the transformant into which exogenous gene is introduced, they have proposed acquiring a uracil-requiring strain (U5 strain) from Cryptococcus sp. S-2 strain by means of spontaneous mutation (See Non-Patent Document 2).
As such, the basidiomycetous yeast such as Cryptococcus genus is very useful as a host for the production of heterologous protein by means of genetic recombination. However, the basidiomycetous yeast has such a problem that large amount of polysaccharides are produced outside the cells as shown in the Non-Patent Document 3. Since the extracellular production of polysaccharides as such raises the viscosity of the culture liquid, it is apt to cause insufficient separation and clogging of ultrafiltration membrane during the steps of cell removal from the culture liquid and of protein purification whereupon purification of proteins in high efficiency has been difficult.
In view of the above, there have been used up to now the methods such as (i) a method wherein the supernatant of the culture liquid is frozen and melted so that the extracellular polysaccharides are aggregated and removed, (ii) a method wherein cold acetone is added to the supernatant of the culture liquid to aggregate the extracellular polysaccharides followed by removing using a glass wool filter and (iii) a method wherein the extracellular polysaccharides are aggregated and precipitated using polyethylene glycol. However, any of those methods is not suitable for its scaling up in view of operability and economy. Thus, with regard to the method (i), there is a problem that freezing of the culture liquid is not easy in big scale. With regard to the method (ii), it is inefficient to pour large amount of acetone thereinto followed by evaporating, and a special consideration is needed for the safety. Further, with regard to the method (iii), there is a risk that the use of polyethylene glycol may deteriorate the ultrafiltration membrane which is used in the latter step and, in addition, there is a problem in the treatment of waste liquid.
Due to those reasons, it is difficult in view of operability, economy and safety to remove the polysaccharides which are produced and secreted in large amount outside the cells. Accordingly, there is a problem that the basidiomycetous yeast is not suitable as a host for the production of heterologous proteins in a large scale.